Zinc-dependent structural stability of human Sonic hedgehog

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Structure and Function

Background:

• Sonic hedgehog (Shh) is a crucial signaling protein involved in embryonic development.
• The N-terminal fragment (ShhN) contains a zinc-binding site essential for its biological activity.
• Understanding the precise role of zinc coordination is vital for elucidating ShhN's mechanism of action.

Purpose of the Study:

• To investigate the role of specific zinc-coordinating residues (H140, D147, H182) and a nearby residue (E176) in the structure and function of human Sonic hedgehog N-terminal fragment (ShhN).
• To determine the impact of zinc coordination on ShhN protein stability and susceptibility to proteolysis.
• To assess the necessity of hydrolase activity for ShhN's in vitro function.

Main Methods:

• Site-directed mutagenesis was used to create alanine mutants of zinc-coordinating residues (H140A, D147A, H182A) and E176A.
• Zinc-binding affinity was quantified using fluorescence-based assays.
• Protein stability was assessed through guanidine hydrochloride denaturation and thermal melting (CD spectroscopy).
• Functional activity was evaluated using established biochemical assays, and proteolysis susceptibility was examined.

Main Results:

• Mutants H140A and D147A showed significantly reduced zinc retention (0.03-0.05 mol/mol) and lower biological activity compared to wild-type ShhN and E176A mutant.
• H140A and D147A mutants exhibited increased susceptibility to proteolysis and reduced structural stability, evidenced by altered thermal denaturation profiles and GuHCl denaturation.
• The E176A mutant was indistinguishable from wild-type ShhN in all tested biophysical and functional assays, suggesting E176 is not critical for zinc site stabilization or ShhN function.
• Zinc binding affinity was reduced in H140A (~1.6 nM) and D147A (~15 nM) mutants compared to wild-type ShhN (

Conclusions:

• The zinc-binding site, particularly residues H140 and D147, is critical for maintaining the structural integrity, stability, and biological activity of ShhN.
• Loss of zinc coordination significantly destabilizes the ShhN protein structure.
• ShhN does not require hydrolase activity for its in vitro biological function, as the E176A mutant, which lacks this potential, remains fully active.